【作者】 房彦军；

【导师】 晁福寰； 高志贤；

【作者基本信息】 中国人民解放军军事医学科学院 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 氯酚类化合物(Chlorophenols, CPs),尤其是五氯酚(Pentachlorophenol, PCP)和三氯酚(Trichlorophenol, TCP)作为廉价、高效的农药主要成分之一,在我国曾被广泛地用于木材防腐、杀菌、灭螺等农业生产。由于CPs残留可以稳定存在于环境中,且可通过呼吸道黏膜,或者皮肤接触等直接或间接方式,进入生物体的血液循环,最后大量蓄积在肝和肾脏当中,造成肝脏、肾脏、免疫及生殖系统的严重损伤,对人类的健康产生严重威胁。但其相关毒性作用及其分子机制尚未阐明。随着现代分子生物学和环境毒理学技术的发展与应用,污染环境与蛋白质组之间相互作用规律的研究,已成为环境蛋白质组学研究领域备受关注的热点问题之一,然而氯酚类化合物对肝脏毒性作用研究,目前还仅限于一些常规毒理学单一指标的分析。本研究以PCP和TCP两种典型氯酚类化合物为研究对象,采用我国特有实验用鱼—稀有鮈鲫(Gobiocypris rarus)为受试动物,以其肝脏蛋白表达谱的变化特征以及肝脏差异表达蛋白的基因转录水平等为切入点,利用比较蛋白质组学方法结合荧光定量PCR等分子生物技术,通过体内和体外暴露实验,从基因调控水平、差异表达蛋白的改变以及终点效应水平等三个方面,探讨CPs对稀有鮈鲫肝脏毒性效应及其分子作用机制,将为氯酚化合物的毒性评价、污染控制和环境监测提供科学依据。本研究主要结果如下:一、本实验利用稀有鮈鲫肝脏细胞原代培养模型,结合噻唑蓝实验(MTT法)和单细胞凝胶电泳(SCGE)方法,探讨在PCP、TCP单独及其复合物暴露条件下,对肝细胞生长存活率的抑制作用及细胞DNA的损伤效应。结果显示,与对照组相比,PCP和TCP单独与复合物暴露均对肝脏细胞正常生长产生明显抑制作用,各暴露组细胞存活率与暴露浓度具有明显的剂量-效应关系。低浓度暴露时(如0.5、2.5μg·L-1)具有显著抑制作用(p<0.05);高浓度暴露时(如25、50μg·L-1)具有极显著抑制作用(p<0.01)。SCGE实验结果显示,与对照组相比,12.5μg·L-1的TCP及复合暴露组均引起DNA显著损伤(p<0.05),12.5μg·L-1、50μg·L-1的PCP暴露组均能引起DNA极显著损伤(p<0.01)。可见,氯酚类化合物对稀有鮈鲫肝细胞正常生长均具有明显抑制作用,且引起其DNA损伤,具有肝细胞毒性。二、本实验将稀有鮈鲫随机分为空白对照组、TCP暴露组(暴露浓度:1、10、100μg·L-1)和PCP暴露组(暴露浓度:0.5、5、50μg·L-1),采用流水暴露系统(28d),电镜观察TCP和PCP暴露致稀有鮈鲫肝脏的组织病理学改变,分析其脏器系数(如性肉和肝肉指数等)的变化;同时,重点利用二维凝胶电泳(2-DE)结合质谱分析技术(MALDI-TOF-MS和TOF-MS-MS),研究TCP和PCP暴露28d后稀有鮈鲫肝脏差异表达蛋白的改变,探讨CPs肝脏毒性效应导致肝脏蛋白质表达谱的变化特征。结果显示,由Mascot软件检索SwissProt数据库鉴定差异蛋白点,共发现74个(其中:PCP和TCP暴露组差异表达蛋白点分别是39个和35个)可能与CPs肝脏毒性效应相关的差异表达蛋白点,通过质谱鉴定技术(MALDI-TOF-MS和TOF-MS-MS)成功鉴定其中29个差异蛋白。利用GO Terms软件,将这些差异表达蛋白功能分为肝脏脂类物质代谢与转运、能量代谢、氧化应激以及其它生物学功能相关蛋白四大类,通过比较发现,CPs暴露后肝脏差异表达蛋白中线粒体ATP合酶和脂肪酸偶联蛋白(FABPs)表达量均发生明显下调,显示氯酚类化合物的肝脏毒性主要干扰脂肪酸转运和代谢、影响线粒体ATP能量合成等生物学过程。同时,CPs暴露致稀有鮈鲫肝脏组织发生明显组织病理学改变,且使肝脏组织的LDH、GST等酶活性升高,SOD活性下降及MDA含量升高,表明CPs暴露导致肝脏细胞氧化应激并产生肝脏毒性效应。三、本实验结合荧光定量PCR技术,分析与暴露致稀有鮈鲫肝脏损伤的肝脏型脂肪酸结合蛋白b(liver-basic fatty acid binding protein b, L-FABP)、谷胱甘肽过氧化酶(glutathione peroxidase, GST)、线粒体ATP合成酶(Mitochondrial ATP synthase, MATPS)、卵黄蛋白原(vitellogenin, Vtg)、果糖-1,6-二磷酸酶(fructose-1,6-bisphosphatase 1b, F-1,6-BP)以及II型肽基精氨酸脱亚氨酸(peptidyl arginine deiminase, type II, PAD)等6个差异蛋白的基因在mRNA表达水平上的变化,探讨CPs暴露所致肝脏损伤效应在其相应基因mRNA表达水平上变化情况,进一步研究PCP和TCP暴露对稀有鮈鲫肝脏损伤效应的分子机制。PCR定量结果显示,与稀有鮈鲫肝脏损伤的主要相关蛋白在mRNA基因表达水平也发生变化,但与差异蛋白表达水平相比较,两者的变化情况存在不一致性。因此,线粒体ATP合成酶、类固醇磺基转移酶和肝脏脂肪酸偶联蛋白等有可能作为,氯酚类化合物致稀有鮈鲫的肝脏毒性效应的蛋白标志物或毒性效应标志物。综上所述,通过建立稀有鮈鲫肝细胞原代培养模型,结合体外暴露实验,证实CPs对稀有鮈鲫肝细胞具有明显毒性效应;体内暴露实验的结果显示,TCP和PCP暴露致稀有鮈鲫肝脏发生组织病理学改变,差异表达蛋白和相关基因mRNA的表达也发生变化,初步阐明CPs肝脏毒性效应的损伤机制主要是通过干扰肝脏脂肪酸转运和代谢、影响线粒体ATP酶能量合成等生物学过程;但差异表达蛋白变化特征和相关基因mRNA表达水平存在不一致性,推断稀有鮈鲫对CPs暴露损伤可能存在着更复杂的基因表达调控机制。更多还原

【Abstract】 The chlorophenol chemicals (CPs) are toxic environmental pollutants that are considered to be ubiquitous, of all the CPs, 2,4,6-trichlorophenol (TCP) and Pentachlorophenol (PCP) had been heavily used to control termites and protect wood from fungal-rot and wood-boring insects throughout the world. The CPs is stable and persistent in the environment, including air, water and soil, and it also can be absorbed into the body by ingestion, inhalation and through the skin, which has been associated with an increased risk of human and rodents carcinogen. In current reports it suggested that residue of PCP in liver and kidney, may enhance toxicity and carcinogenicity of PCP, since it is capable of inducing oxidative damage,and other results indicated that the exposure to rats at high levels of PCP will lead to increase the body temperature, liver defects, damage to the immune system and reproductive defects, while severe exposure of PCP could cause an acute and occasionally fatal illness mainly due to PCPs cytotoxicity, but the correlated molecular damage mechanisms has not been clear clarification.Recently, with the development of molecular biology and it’s application in the environmental toxicology research,to research the principles and techniques for protein expression under polluted environment have long been of concern. Previous studies have mostly focused on the single toxicity respect of these chemicals and there is rarely report so far about the effect on global protein profiles exposed to CPs.Taking Gobiocypris rarus as the test animals, this study was designed to investigate the hepatotoxicity of chlorophenols on fish in genetic transcription, protein and enzyme activity levels from both in vitro and in vivo respectively. And this dissertation is to discuss the molecular mechanism of the toxic effect following the CPs exposure, the toxicoproteomic approach and the real–time PCR analysis method were applied to identify proteins differentially expressed in the livers of rare minnow. The results will provide useful data for not only the assessment of the potential risk of Chlorophenols, but also the understanding of their mechanism of action. The main results are as follows:1. The system of primary cultural hepatocytes from rare minnow was successfully established, and the hepatocyte of rare minnow were also extracted for primary culture, whereby the inhibition rate and DNA damage of hepatocyte exposed to these materials were examined by MTT assay and single-cell gel electrophoresis (SCGE), respectively. Results show that: Each group observed a inhibition effect on hepatocyte, furthermore, in each group, the low-dose exposure (0.5, 2.5μg·L-1) showed a obvious dose-effective and a significant inhibition effect compared with control group when reaching medium and high dose (25, 50μg·L-1). The SCGE results show that is a significant increase in DNA damage at PCP exposed dose of 12.5, 50μg·L-1 (p<0.01), TCP and joint exposure dose of 12.5μg·L-1 (p<0.05). There is significant (p<0.01, p<0.05) difference between the damage rate of these treated groups and that of control group, suggesting that the PCP and TCP have cytotoxicity and can induce the hepatocyte DNA strand to break in vitro.2. For further understanding of mechanisms of action and identify the potential protein biomarkers for CPs exposure, two-dimensional electrophoresis coupled with mass spectrometry (MALDI-TOF-MS) was used to identify the differentially expressed proteins in the livers of rare minnow following TCP exposure of 1, 10, 100μg·L-1, and PCP exposure of 0.5, 5, 50μg·L-1 under flow-through conditions for period time of 28d, respectively. The pathological of liver was associated with a hypertrophy of hepatocytes and damages to cellar structure by pathological observation, and the tissue somatic indices were also analysis.After comparison of the protein profiles between treated and control groups, 39 and 35 protein spots were found altered in abundance (>2-fold) from PCP and TCP treated groups, respectively. Matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (MALDI-TOF-MS) analysis allowed the unambiguous identification, and the 30 protein spots were identified successfully. The identified proteins were then matched to specific biological processes or functions by searching Gene Ontology (GO Terms) using Uniprot/Swissprot database and submitted to ingenuity, we classified those proteins manually to a variety of cellular biological processes, according to that took part in a variety of cellular biological processes such as: transport, metabolism of lipid, response to oxidative stress, protein repair, oxidative phosphorylation and other related processes. And the results show that the gonadosomatic index (GSI) was significantly greater than that of control, while, the hepatosomatic index (HSI) for female and male contained statistically differences compared to control groups. PCP increased LDH and GST activity in liver of Gobiocypris rarus, and decreased SOD activity, these results indicate that exposure to Chlorophenols may lead to generation of free radicals, depletion of antioxidants and peroxidation of lipids, thus resulting in oxidative stress in liver of the Gobiocypris rarus. 3. The transcriptional analysis of six mRNA encoding proteins altered by PCP exposure in the proteome analysis was determined by real-time PCR, such as Mitochondrial ATP synthase, Steroid Sulfotransferase-like protein, Liver-basic fatty acid binding protein b and Vitellogenin were observed to be potential biomarker in toxicological studies of rare minnow following PCP exposure, which should be the main focus of studies on mechanism of PCP toxicity in the future.In conclusion, the system of primary cultural hepatocytes from Gobiocypris rarus was successfully established. The in vitro analysis using the primary cultural hepatocyte was carried out to confirm that the PCP and TCP have cytotoxicity can induce the hepatocyte DNA strand to break. Based on the observed histopathological changes, and the differentially expressed proteins and the mRNA level in the livers of Gobiocypris rarus following TCP and PCP exposure in vivo, we can concluded that the molecular damage mechanisms of toxicity effect of CPs mainly by the interfering the fatty acid transporting, the mitochondrial ATP synthase and the related fundamental regulation processes. In addition, the discrepant results between mRNA and protein levels suggested that complicated regulatory mechanisms of gene expression were implicated in the response to chlorophenols exposure.更多还原